Laryngeal Tissue Engineering using Rabbit Adipose Derived Stem Cells in Fibrin: A Pre-Clinical Model

Vocal fold scarring is a clinical problem without reliable treatment. Tissue engineering of a vocal fold replacement is an exciting potential treatment for vocal fold scars that involve multiple layers of the vocal fold. Human adipose-derived stem cells (ASC) were previously used to produce a promising vocal fold cover layer replacement. However, relevant in vivo studies are needed before human application, and implanting the human cells in animal larynges would introduce significant risk and data confounding. We therefore report here the development of a construct based on rabbit ASC with the potential for use in pre-clinical implantation studies. Rabbit ASC were isolated and cultured in a three-dimensional fibrin matrix to create an implantable construct resembling the vocal fold mucosa. Key differences between the human cell and the rabbit cell models are highlighted. DOI : 10.14302/issn.2379-8572.joa-14-611 Corresponding Author: Dr. Jennifer Long Department of Head and Neck Surgery UCLA Medical Center, 10833 Le Conte Ave, CHS 62-232 Los Angeles, CA 90095-1624 jlong@mednet.ucla.edu ; Ph: 310.825.3015 ; Fx: 310.794.7083


Translating in vitro models
In this work, we set out to develop a tissueengineered construct suitable for implantation in rabbit vocal folds. Rabbits are a favorable animal model for study of the larynx due to adequate laryngeal size for implantation surgery, histologic similarities to human larynges, and economical animal husbandry relative to larger mammals 7 . They can undergo phonation both in vivo and in excised larynx preparations 8,9 . Furthermore, minimum of 60 minutes with agitation. The solution was then added to "Control Medium" consisting of Dulbeco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), 1% amphotericin B, and 1% penicillin/ streptomycin in a 1:1 ratio to halt digestion and centrifuged at 1600rpm for 5 min. The resulting stromovascular fraction pellet was collected from beneath the fat-laden supernatant, and distributed for culture in control medium on standard tissue culture plates. The cultures were incubated at 37 o C with 5% CO 2 and medium was changed twice a week until confluent. Confluent cells were passaged or harvested for use on or before the third passage.

Rabbit ASC differentiation
Rabbit ASC in culture were harvested using trypsin-EDTA and re-seeded in 12-well culture plates.

Fibrin-ASC constructs
Human cryoprecipitate, rabbit fibrinogen, and bovine fibrinogen were investigated as fibrin sources.

Pluripotency of rabbit stromovascular fraction cells
Light microscopy demonstrated that cultures induced to differentiate into adipogenic and osteogenic cell types showed morphological changes after 2 weeks in culture (Fig. 1). After 28 days in culture, induced cells showed positive staining for lipid after adipogenic culture       with the rabbit fibrinogen constructs, for the most relevance for future rabbit implantation.
For the constructs made with rabbit fibrinogen, hematoxylin and eosin microscopy showed a fibrin lattice with similar gross morphology to the vocal fold lamina propria and epithelium. Cell nuclei were identified throughout the construct (Fig. 4). There was no observable morphologic difference between constructs treated with EGF and those that were not.

Differentiation of rabbit ASC in fibrin gel culture
Spontaneous differentiation to mesenchymal phenotypes did not occur. Histological staining using Oil Red O and Von Kossa on tissue-engineered constructs in cultured for 14 days, with and without EGF treatment, showed negative staining for lipid and mineral (Fig. 5).

Epithelial differentiation was not induced by EGF
and an air interface in this rabbit ASC culture, as

DISCUSSION
Tissue engineering and regenerative medicine hold promise for diseases that are currently difficult to treat because of lack of suitable therapies or self-repair mechanisms. Vocal fold scarring is an excellent example.
The specialized extracellular matrix of the vocal fold lamina propria and its attached epithelium are, thus far, irreplaceable after injury. Its geometry also makes the vocal fold mucosa a good candidate for tissue engineering in the laboratory. It is a small, thin structure with low metabolic activity due to sparse cellularity; these factors ensure adequate nutritional perfusion without need for neovascularization.
Befitting the label "stem cells", adipose-derived stem cells possess the capabilities of self-renewal and multiple differentiation potential 11 . ASC may be more clinically applicable than other stem cell types because they are quite easily harvested from adult liposuction material and are simple to maintain in culture 12 . As such, ASC have been proposed as a cell source for engineering and repair of nearly any conceivable tissue type [13][14] .
They are currently being investigated in nearly one Rabbit ASC have been shown to differentiate into an epithelial phenotype when exposed to an air liquid interface and a growth factor cocktail, consisting of all-trans retinoic acid, EGF, and further enhanced by hepatocyte growth factor and keratinocyte growth factor for 12 days 18 . That study was not performed in a three-dimensional scaffold.
Fibrin was chosen as the biological scaffold for this study due to its importance in normal wound healing, its biodegradable nature, and its potential for autologous sourcing from plasma. As the principal and increase elastin production, vocal fold smoothness and collagen organization 9, 21-24 . Furthermore, adding a micronized acellular dermal matrix or hydrogel scaffold to the injection decreased scarring 9,25 . The wound healing properties of ASC may be adequate to prevent scar formation after implantation, while the fibrin scaffold may further support ASC survival and differentiation. These aspects will be tested in a rabbit implantation trial.

CONCLUSION
Rabbit adipose-derived stem cells were used to create a three-dimensional tissue-engineered construct suitable for vocal fold cover replacement. The construct